The goal of our research program is to understand the mechanisms of regulation of biological activity in macromolecular complexes through structural analysis, using X-ray crystallography as our primary tool. (1) Adeno-associated virus (AAV) Rep. Only one animal virus, AAV, integrates its genome into a particular location in human chromosomal DNA. This unique property has important implications for the targeted delivery of genes in the context of gene therapy vectors. Viral integration requires the AAV Rep protein, a multifunctional enzyme that possesses site-specific DNA binding activity, endonuclease activity, ATPase activity, and 3'-to-5' helicase activity. To shed light onto the mechanism of integration, we have previously determined the structures of the endonuclease domain of AAV5 Rep bound to two specific DNA regions found within the three-way DNA junction that marks the end of the viral genome. We are now extending this work to investigate the properties of larger protein-DNA complexes formed by the full-length Rep protein. (2) DNA transposition. We are interested in the wide variety of solutions that mobile DNA elements have devised to move DNA from one location to another. We are currently investigating several systems including Hermes, a eukaryotic transposon from Musca domestica, and ISHp608, an insertion sequence originally isolated from Helicobacter pylori. Hermes serves as a model system for the widely distributed hAT family of transposons, representatives of which are found in fungi, plants, and animals including vertebrates. We have recently determined the structure of an active fragment of the Hermes transposase, the only structure reported to date for any eukaryotic DNA transposase. Unlike the characterized prokaryotic transposases, the Hermes transposase forms hexamers in solution, and we have been able to use our experimentally determined crystal structure to model the interfaces that are likely important for this unusual assembly. ISHp608 provides an example of yet another transposition system with unusual features: it transposes using a different mechanism from that of other known transposons as one of the identified reaction intermediates is a circular double-stranded version of the insertion sequence in which the ends are precisely joined to one another. Furthermore, the primary sequence of its transposase, TnpA, does not contain any of the amino acid motifs that would place it within any of the known transposase families. We have recently determined the structure of TnpA alone and in complex with a specific DNA sequence that is located close to each end of the insertion sequence. It appears likely that high affinity binding of this 26-bp sequence, which forms a DNA stem-loop with a single extrahelical base, is the mechanism by which the dimeric TnpA is able to bring together both ends of the insertion sequence and coordinate the chemical reactions that are necessary to move the DNA segment from one place to another.